This invention relates to nucleic acid and amino acid sequences of human transferases and to the use of these sequences in the diagnosis, treatment, and prevention of autoimmune/inflammatory, neurological, reproductive, and gastrointestinal disorders and cancer.
Transferases, enzymes that catalyze group transfer reactions, are classified by the type of group transferred. One-carbon groups are transferred; for example, methyltransferases transfer methyl groups from S-adenosyl-methionine to substrates. Nitrogenous groups are transferred; for example, aminotransferases transfer amino groups. Other groups transferred include aldehyde or ketone, acyl, glycosyl, alkyl and aryl other than methyl, phosphorus-containing, sulfur-containing, and selenium-containing groups.
The enzyme glutamine-phenylpyruvate aminotransferase, also known as glutanaine transaminase K (GTK), catalyzes several reactions with a pyridoxal phosphate cofactor. GTK catalyzes the reversible conversion of L-glutamine and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine. L-methionine, L-histidine, and L-tyrosine can substitute for L-glutamine in this reaction. GTK also catalyzes the conversion of kynurenine to kynurenic acid. Kynurenic acid, a tryptophan metabolite, is an antagonist of the N-methyl-D-aspartate (NMDA) receptor in the brain and may exert a neuromodulatory function. Alteration of the kynurenine metabolic pathway may be among the causative factors leading to several neurological disorders. GTK also possesses cysteine conjugate xcex2-lyase activity which is involved in the metabolism of halogenated xenobiotics conjugated to glutathione. GTK action on the cysteine conjugates of xenobiotics yields metabolites that are nephrotoxic in rats and neurotoxic in humans. The neurotoxicity may be related to the kynurenine aminotransferase activity of GTK. GTK is expressed in kidney, liver, and brain. Both cytosolic and mitochondrial forms exist. Human and rat GTK genes have been isolated which encode proteins of 422 and 423 amino acids respectively. Both human and rat GTKs contain a putative pyridoxal phosphate binding site. (ExPASy ENZYME: EC 2.6.1.64; Perry, S. J. et al. (1993) Mol. Pharmacol. 43:660-665; Perry, S. et al. (1995) FEBS Lett. 360:277-280; and Alberati-Giani, D. et al. (1995) J. Neurochem. 64:1448-1455.)
The enzyme kynurenine/a-aminoadipate aminotransferase (AadAT) catalyzes two reactions with a pyridoxal phosphate cofactor. AadAT catalyzes the reversible conversion of xcex1-aminoadipate and xcex1-ketoglutarate to xcex1-ketoadipate and L-glutamate. This conversion is involved in lysine metabolism. AadAT also catalyzes the transamination of kynurenine acid to kynurenic acid. As described above, kynurenic acid is an NMDA receptor antagonist. Both soluble and mitochondrial forms of AadAT have been purified. A soluble AadAT is expressed in rat kidney, liver, and brain. The rat AadAT nucleotide gene encodes a protein of 425 amino acids which contains a putative pyridoxal phosphate binding site. (Nakatani, Y. et al. (1970) Biochim. Biophys. Acta 198:219-228; Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335.)
Protein-arginine methyltransferases catalyze the posttranslational methylation of arginine residues in proteins, resulting in the mono- and dimethylation of arginine on the guanidino group. Known substrates are histones, heterogeneous nuclear ribonucleoproteins (hnRNPs), and myelin basic protein. This otherwise unusual posttranslational modification is common in hnRNPs and may regulate their function. hnRNPs function in the nucleus in mRNA processing, splicing, and transport into the cytoplasm. Homologous protein-arginine methyltransferases that methylate hnRNPs have been cloned from yeast, rat, and man. These protein-arginine methyltransferases contain five sequence motifs, termed region I, post-region I, region II, region III, and post-region III, that may be involved in binding S-adenosyl-methionine. One human gene (HRMT1L1) encodes a 433 amino acid protein. The other human gene (HRMT1L2) may be alternatively spliced to yield three protein-arginine methyltransferases, of length 343, 347, and 361 amino acids respectively, with different amino termini. The protein encoded by the cloned rat protein-arginine methyltransferase gene (PRMT1) interacts with the TIS21 protein and the homologous BTG1 protein. The intermediate-early TIS21 protein is the product of a gene induced by treatment of cells with mitogens such as epidermal growth factor, and the-BTG1 protein is the product of a human gene located near a chromosome translocation breakpoint associated with chronic lymphocytic leukemia. The HRMT1L2 protein interacts with the cytoplasmic domain of the interferon receptor. This interaction suggests that protein methylation may be an important signaling mechanism for cytokine receptors (Lin, W.-J. et al..(1996) J. Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H. S. et al. (1998) Genomics 48:330-340.)
The discovery of new human transferases and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of autoimmune/inflammatory, neurological, reproductive, and gastrointestinal disorders and cancer.
The invention features substantially purified polypeptides, human transferases, referred to collectively as xe2x80x9cHUTRANxe2x80x9d and individually as xe2x80x9cHUTRAN-1xe2x80x9d, xe2x80x9cHUTRAN-2xe2x80x9d, and xe2x80x9cHUTRAN-3.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (SEQ ID NO: 1-3) and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1-3 or to fragments of any of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID) NO:4, SEQ ID NO:5, and SEQ ID NO:6 (SEQ ID NO:4-6), and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:4-6 and fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:4-6 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof, as well as a purified agonist and a purified antagonist to the polypeptide. The invention also provides a method for treating or preventing an autoimmune/inflammatory disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides a method for treating or preventing a neurological disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides a method for treating or preventing a gastrointestinal disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an, antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-3 and fragments thereof to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the method further comprises amplifying the polynucleotide prior to the hybridizing step.